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Abstract

Introduction: Proof-of-concept has been provided regarding biological pacemakers based on HCN2, but basal and maximal rates are less than ideal. We previously reported that co-expression of HCN2 and the Ca2+-stimulated adenylyl cyclase 1 (AC1) in cultured ventricular myocytes results in elevation of intracellular cAMP and faster beating rate than HCN2 plus GFP or HCN2 plus AC6. In the present study we asked if this also occurred in vivo and if it was entirely explained by the effect of cAMP on expressed HCN2.

Methods: The adenoviral constructs of HCN2 and AC1 or GFP were implanted into the left bundle branch of AV-blocked dogs to test in vivo biological pacemaker function. To establish HCN2 dependent and AC subtype specific effects on pacemaker function, we co-expressed HCN2/RE (cAMP insensitive) with GFP, AC1 and AC6 in neonatal rat ventricular myocytes.

Results:In vivo, we found that co-expression of HCN2 plus AC1 compared to HCN2 plus GFP results in faster escape rates which could be increased still further by epinephrine (155% increase at 1 μg/kg/min) and faster maximal rates (see figure). Hence the prior demonstration of in vitro efficacy was replicated in vivo. In cell culture, replacing HCN2 with HCN2/RE - to eliminate cAMP binding to the expressed channel - did not prevent AC1 from enhancing rate compared to AC6 or GFP controls (HCN2/RE+AC1 210±22 bpm, P<.05 vs HCN2/RE+GFP, 160±15 & HCN2/RE+AC6, 125±22 bpm). The increased rate in the AC1 group occurred with no effect on HCN2/RE current indicating that the outcome at least in part reflects actions on endogenous targets of cAMP.